| Objective:To explore the effect of intraperitoneal injection of serum exosome of normal mice after acupuncture(Acupunctuer-exosome,Acu-exo)on the survival time of hypoxic mice and the possible target of its effect,so as to provide experimental basis for the role of exosome in acupuncture.Methods:Research content 1 : Observation and study on the survival time of hypoxic mice by electroacupuncture pretreatmentat Zusanli Point.1.This experiment was repeated twice.In the first experiment,32 normal KM male mice were randomly divided into electroacupuncture group(n = 16)and control group(n =16).Electroacupuncture pretreatment was performed with acupuncture(0.25×13 mm)into Zusanli(3-5mm).The electroacupuncture parameters were as follows: frequency 10 Hz,density wave,time 15 min,intensity 5.Immediately after electroacupuncture,the mice were put into the 250 m L jar containing 30 g sodium lime to create anoxic environments.Taking the survival time of mice as the observation index.The control group only replicated the model of hypoxia without acupuncture.In the second experiment,30 normal KM male mice were randomly divided into control group(n = 15)and electroacupuncture group(n =15).The rest of the operation is the same as the first experiment.Research content 2: Observation and study on the effect of Acu-exo on the survival time of hypoxic mice.1.Extraction and Identification of Acu-exo in normal mice after electroacupuncture at Zusanli Point for different days: 32 normal KM male mice were randomly divided into control group(E0),electroacupuncture 1-day group(E1),electroacupuncture 4-day group(E4)and electroacupuncture 7-day group(E7),with 8 mice in each group,frequency: once a day,parameters were the same as before.Eyeball blood was taken from each group after acupuncture,and Acu-exo,was extracted from serum by Exo Quick method.The exosome body size,shape and markers were identified by transmission electron microscope,nanoparticle tracking analysis and Western blotting.2.Observation and study on the survival time of hypoxic mice by injection of solvents PBS and DMSO : 18 normal KM male mice were randomly divided into control group(n=6),PBS group(n=6)and DMSO group(n=6).The normal mice in the PBS group were injected intraperitoneally with aseptic PBS(0.1m L/10g),and the hypoxia model was replicated 0.5 hours later,while in the DMSO group,the normal mice were injected intraperitoneally with DMSO(concentration of 0.2%,0.1ml/10g),and the hypoxia model was replicated after 1.25h,and the survival time of the mice was used as the observation index.3.Observation and study on the survival time of hypoxic mice infused with Acuexo: 50 normal KM male mice were randomly divided into control group,E0 exosome group,E1 exosome group,E4 exosome group and E7 exosome group with 10 mice in each group.The exosomes of each group were infused,that is,the exosomes of E0,E1,E4 and E7 were extracted in experiment 1.Each group was injected intraperitoneally with Acu-exo(4mg/kg,0.1ml/10 g,).After 0.5h later,the hypoxia model was replicated and the survival time of mice was used as the observation index.4.Observation and Researchon on the effect of antagonistic circulating exosome on the survival time of hypoxic mice: 40 normal KM male mice were randomly divided into control group(n=10),electroacupuncture group(n=10),electroacupuncture+DMSO group(n=10)and electroacupuncture+GW4869 group(n=10).Electroacupuncture intervention was performed in the electroacupuncture group.Acupuncture(0.20-0.10)was used to acupuncture into Zusanli point(3-5mm).The electroacupuncture parameters were as follows: frequency 10 Hz,density wave,time 15 min,intensity 5.The mice in the electroacupuncture+DMSO group and the electroacupuncture+GW4869 group were injected intraperitoneally with DMSO(concentration of 0.2%,0.1ml/10g)and GW4869(dissolved in DMSO with exocrine antagonist GW4869,3mg/ml,0.1ml/10g)respectively.One hour later,the mice were pretreated with electroacupuncture,and the electroacupuncture parameters were the same as before.The hypoxia model was replicated immediately after electroacupuncture intervention,and the survival time of mice was taken as the observation index.Research contents 3:Complex network analysis of the mechanism of Acu-exo improving the survival time of hypoxia.1.The type and content of proteins carried by serum exocrine extracted from the control group and the normal acupuncture group were measured by non-target quantitative proteomics technique(Label Free).The acupuncture significant difference proteins with a change multiple of more than 1.5 and p<0.05.263 normal acupuncture significant difference proteins were selected as source data.Significant differential proteins were transformed into human genes by Uniport protein database and human genome database by HGNC.The human genes related to hypoxia were screened from OMIM database,Dis Ge NET database and Genecards database,and the common target genes were screened by the intersection of the two.Then use DAVID database to establish KEGG pathway and GO functional database of common targets.construct significant differential target-KEGG pathway network connection diagram.The significant difference target-KEGG pathway network connection diagram,significant difference target-KEGG pathway network connection diagram,significant difference target-GO functional network connection diagram and bubble diagram were constructed by Gephi software,and the data mining algorithms such as connection indegree,connection outdegree and other running algorithms were used to screen the anti-hypoxia targets of serum exosome(Acu-exo)which might be effective after acupuncture.Results:1.Electroacupuncture pretreatment can improve the survival time of hypoxic mice.After electroacupuncture pretreatment,the hypoxia model was replicated twice,the first experiment was repeated,the survival time of the blank control group was 32.61±2.96 min,and the survival time of the electroacupuncture group was 50.84±3.89 min.In the second experiment,the survival time of the blank control group was 37.73 ±4.00 min,and that of the electroacupuncture group was 50.35±6.35 min.The results of the two repeated experiments were simila.the survival time of the electroacupuncture group was significantly higher than that of the control group(P < 0.01).2.The morphology,size and markers of exocrine bodies were in accordance with the requirements and standards identified by extracellular vesicle association.The Acu-exo extracted from control group(E0),electroacupuncture 1-day group(E1),electroacupuncture 4-day group(E4)and electroacupuncture 7-day group(E7)were determined for BCA protein concentration.TThe total protein concentrations of E0,E1,E4 and E7 were respectively 9.19mg/m L,11.22mg/m L,10.74mg/m Land 15.69mg/m L.Transmission electron microscopy showed that the diameter of the exocrine body was ≥ 30 and ≤ 200 nm.Among them,the serum exosome were in the shape of round or round-like vesicles,or distributed individually,or clustered in groups.The tracking analysis of nanoparticles showed that the detection reports of E0,E1,E4 and E7 groups showed that the MODE curve was linear and smooth,suggesting that there were few impurities,and the particle size peaks were 67.6nm,103.3nm,72.4nm and 137.9 nm,respectively.The peak particle size was consistent with the theoretical exosome size(30-200nm).The protein spectrum of exosome in group E7 was analyzed by SDS-PAGE electrophoresis.The electrophoresis showed that the protein bands CD63,CD9,TSG101 and Clanexin met the criteria,suggesting that the extraction of exosome was not disturbed by miscellaneous proteins,and the purification effect was good.3.The solvents PBS and 0.2%DMSO had no significant effect on the survival time of hypoxic mice.In the control group: 33.25±2.38 min;PBS group: 34.22±3.19 min;DMSO group: 37.55±5.69 min.There was no significant difference in the survival time of hypoxic mice among control group,PBS group and DMSO group.4.Intraperitoneal infusion of Acu-exo could improve the survival time of hypoxia-tolerant mice.33.29±2.84 min in the control group,34.99±3.59 min in the E0 exocrine group,38.00 ±4.22 min in the E1 exocrine group,39.59±3.77 min in the E4 exocrine group,and 40.944±2.70 min in the E7 exocrine group.The hypoxia tolerance time of E1 exocrine transfusion group,E4 exocrine transfusion group and E7 exocrine transfusion group was significantly different from that of the control group(p < 0.01).The hypoxia tolerance time of the control group was not significantly higher than that of the E0 exocrine group(p > 0.05).The hypoxia tolerance time of mice in E1 exocrine group,E4 exosome group and E7 exosome group increased compared with each other.5.GW4869 antagonizing circulating exosomes reduced the efficacy of electroacupuncture in improving survival time of hypoxic mice.In control group: 29.88±2.35min;electroacupuncture group: 46.23±4.99min;electroacupuncture+DMSO group:46.97 ± 4.76min;electroacupuncture+GW4869 group:31.41 ± 3.36min;compared with electroacupuncture+GW4869 group,the survival time of acupuncture group was significantly higher than that of electroacupuncture+GW4869 group.Compared with the control group,the hypoxia tolerance time of the electroacupuncture group was significantly higher than that of the control group(p < 0.01),but there was no significant difference between the electroacupuncture+GW4869 group and the control group.6.Epidermal growth factor receptor(EGFR),thrombospondin-1(THBS1)and coagulation factor II(F2)carried by Acu-exo may be effective targets for acupuncture tolerance to hypoxia.There are 221 genes mapped to human by significant differences in serum exosome proteins between the normal electroacupuncture group and the control group.There are 6104 human genes related to hypoxia in OMIM database,Dis Ge NETs database and Genecard database.There were 114 genes in common between the two.Complex network analysis showed that the possible effective targets of serum exosome extracted after acupuncture are epidermal growth factor receptor(EGFR),thrombospondin-1(THBS1)and coagulation factor II(F2).Conclusion:1.Acu-exo can improve the survival time of hypoxic mice,and there is a certain doseeffect rule.2.Exocrine plays an important role in electroacupuncture to improve the survival time of hypoxic mice.3.EGFR,THBS1 and F2 carried by exosomes may be effective target substances of electroacupuncture against hypoxia. |
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